Neurotrophic Activity and Its Modulation by Zinc Ion of a Dimeric Peptide Mimicking the Brain-Derived Neurotrophic Factor N-Terminal Region

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) essential for neuronal development and synaptic plasticity. Dysregulation of BDNF signaling is implicated in different neurological disorders. The direct NT administration as therapeutics has revealed to be challenging. This has prompted the design of peptides mimicking different regions of the BDNF structure. Although loops 2 and 4 have been thoroughly investigated, less is known regarding the BDNF N-terminal region, which is involved in the selective recognition of the TrkB receptor. Herein, a dimeric form of the linear peptide encompassing the 1–12 residues of the BDNF N-terminal (d-bdnf) was synthesized. It demonstrated to act as an agonist promoting specific phosphorylation of TrkB and downstream ERK and AKT effectors. The ability to promote TrkB dimerization was investigated by advanced fluorescence microscopy and molecular dynamics (MD) simulations, finding activation modes shared with BDNF. Furthermore, d-bdnf was able to sustain neurite outgrowth and increase the expression of differentiation (NEFM, LAMC1) and polarization markers (MAP2, MAPT) demonstrating its neurotrophic activity. As TrkB activity is affected by zinc ions in the synaptic cleft, we first verified the ability of d-bdnf to coordinate zinc and then the effect of such complexation on its activity. The d-bdnf neurotrophic activity was reduced by zinc complexation, demonstrating the role of the latter in tuning the activity of the new peptido-mimetic. Taken together our data uncover the neurotrophic properties of a novel BDNF mimetic peptide and pave the way for future studies to understand the pharmacological basis of d-bdnf action and develop novel BDNF-based therapeutic strategies.

S3 one-way ANOVA with Dunnett's multiple comparison test, # p<0.05 versus BDNF, according to one-way ANOVA with Sidak's multiple comparison test).     Tables   Table S1. Main H-bonds observed in the binding of TrkB/d-bdnf, indicating the amino acid residues participating to the non-covalent interactions.

Preparation of hBDNF protein
The preparation of mature hBDNF has been recently described in detail 1 . Briefly, BL21(DE3) E. coli were transformed with hproBDNF construct in pET11a plasmid, and protein expression induced with 1 mM of isopropyl-b-thio-galactoside (IPTG). After 5 hours bacteria were collected by centrifugation, and the pellet resuspended with Lysis Buffer (10 mM TRIS HCl pH 8, 1 mM EDTA and 1 mg/ml lysozyme), incubated at room temperature for 1 h, sonicated on ice, and incubated again with a 3 mM

Peptide synthesis and purification
The peptides HSDPARRGELSV-NH2 (m-bdnf) and the scrambled sequence SRAGPHLRDVSE-NH2 (s-bdnf) were assembled using the solid phase peptide synthesis strategy on a Pioneer Peptide S10 Synthesizer. All amino acid residues were added according to the TBTU/HOBT/ DIEA activation method for Fmoc chemistry on a NovaSyn-TGR resin (loading 0.18mmolg 1, 0.33mmol scale synthesis). Removal of Fmoc protection was achieved by means of a 20% piperidine solution in DMF.   Densitometric analysis of the obtained bands was done with ImageJ software (https://imagej.nih.gov/ij/) or ImageLab software (Bio-Rad).

Phosphorylation of TrkB and downstream signaling effectors
ERK and AKT phosphorylation were assessed in a 96-well plate format, performing an in cell ELISA assay as previously described 4 . SH-SY5Y or HEK 293T cells were seeded at a density of 5.000 and 80.000 cell/well in 96 multiwell respectively, and the former were differentiated as describe above. (1:100, Thermofisher) in 2.5% BSA/PBS for 2h at room temperature. Coverslips were then mounted with Fluoroshield-DAPI (Sigma-Aldrich) and imaged with the laser scanning confocal microscope as described in the previous section, using a 20× air objective (NA 0.75) and pinhole set to 1 AU.
Images were acquired at 1024 pixel × 1024 pixel resolution using a 405 nm laser (emission window 400-500 nm) and a 488 nm laser (emission window 475-575 nm). The acquired images were analysed using FIJI ImageJ software. β3Tubulin positive cells were identified in each field by visual inspection and normalised on the total cells counted in the DAPI channel.

RT-PCR analysis of SH-SY5Y differentiation
At the end of each differentiation treatment and relative controls, cells were collected, and total RNA was extracted using the Rneasy Mini Kit (Qiagen). The RNA concentration and purity were checked  Table S4. HPRT1 and NEFM primers were purchased by Biorad (qHsaCID0016375 and qHsaCED0043021, Bio-Rad). The mRNA levels for each sample were normalized against GAPDH and HPRT1 mRNA levels, and the relative expression was calculated by using the methods of the ΔΔCt value.

Peptide Simulation Details
The initial coordinates of d-bdnf connected through a disulfide bridge have been obtained using CHIMERA software 5 . Those coordinates have been energy minimized through 500000 Steepest Descent followed by 28 ns of equilibration with standard molecular dynamics using the amber99SB force field 6 16 . The TIP3P potential 7 has been used for water solvent molecules in a simulation box of 6.0 x 6.0 x 6.0 nm3. The MD simulation has been performed by using GROMACS MD code -version 2020.2 8 .

S15
Far-UV CD spectra were obtained at 298 K under a constant flow of nitrogen by using a Jasco model 810 spectropolarimeter. Measurements were carried out in water by using 1 cm path length cuvettes in the range =200-260 nm. All solutions were freshly prepared with doubly distilled water. Far-UV CD spectra were acquired by using Zn2+ and peptide concentrations ranging from 2.0x10 -6 to 2.0x10 -5 M.

Potentiometric measurements
Potentiometric titrations were carried out on a Titrando 905 automatic, using a combined glass-

Statistical Analysis
The Graph-Pad Prism program (GraphPad Software Inc., San Diego, CA, United States) was used for data analysis and graphic presentation. Statistical analysis was performed by ordinary one-way analysis of variance (ANOVA) with one way Bonferroni post-hoc, or t-Test, with details indicated in the relative figure captions. All data values were reported as the mean of the values obtained in at least two independent experiments. P ≤ 0.05 was considered statistically significant.